2. 38 M.M. Hernandez et al. / Journal of Ethnopharmacology 67 (1999) 37–44
´
also used as an anti-inflammatory agent, while secticidal) performed with crude extracts from
V. gaumeri is used to treat colds and coughing different plant parts of V. trifolia.
spells (Ekundayo et al., 1990; Chawla et al.,
1992; Argueta et al., 1994; Damayanti et al.,
1996). It is well known that a considerable num- 2. Materials and methods
ber of plant species, besides their popular use as
medicine in many countries, possess insecticidal 2.1. Plant material
activities. The genus Vitex sp. is not an excep-
tion. V. negundo has larvicidal activity against Leaves and stems of V. trifolia L. (Verbenaceae)
the mosquito species Culex quinquefasciatus and were collected at Poblado ‘El Cinco’ (El Carrizo
Valley, Sinaloa, Mexico). A specimen was
Anopheles stephensi (Pushpalatha and Muthukr-
deposited at the IMSSM Herbarium, Mexico City,
ishnan, 1995), and acts as a deterrent to the
voucher number 11878. Collected material was
mosquito Aedes aegypti (Hebbalkar et al., 1992).
dried in the dark at room conditions. Later, both
V. rotundifolia also shows deterrent properties
leaves and stems were extracted by maceration in
towards A. aegypti (Watanabe et al., 1995). Sev- hexane during 72 h in darkness. Residuals were
eral other Vitex species are currently being in- further extracted with dichoromethane (DCM)
vestigated in specific programs of pest control following the same procedure. Final extraction was
(Rahman and Bhattacharya, 1982; Epila and performed with methanol. The plant extracts were
Ruyooka, 1988; Sudarsanam et al., 1995). then evaporated under reduced pressure and
V. trifolia has been reported to have both prepared to perform the assays.
medicinal and insecticidal properties. It alleviates
pain derived from rheumatism and sprained 2.2. Cytotoxicity assays
joints when applied topically. Also, the leaves
are used to treat intermittent fever, while the All cell lines representing cervix carcinoma
tiny flowers are administered as an infusion to (SQC-1 UISO), ovarian cancer (OVCAR-5), colon
treat fever accompanied by vomiting and severe carcinoma (HCT-15 COLADCAR) and human
thirst (Ramesh et al., 1986; Thein et al., 1995). nasopharyngeal carcinoma (KB) were maintained
An insect antifeeding activity has been recorded on RPMI medium supplemented with 10% fetal
for seeds (Hosozawa et al., 1974). There have bovine serum (FBS), and incubated at 37°C in an
been various studies on the chemical structures atmosphere of 5% CO2 in air (95% humidity). The
of compounds isolated from both V. trifolia cells at a log phase of their growth cycle were
leaves and fruit extracts. The isolation of treated in triplicate with various concentrations of
flavonoids: casticin, 3,6,7-trimethyl quercetagetin the extracts (1, 10, 100 mg/ml) dissolved in 20%
(Zeng et al., 1996), vitexin, artemetin, 5-methyl dimethylsulfoxide (DMSO) in water. Initial cell
suspensions which contained 25 000 cells/ml
artemetin, 7-desmethyl artemetin, luteolin (Nair
(counted on a hemocytometer), were incubated
et al., 1975), luteolin-7-O-b-D-glucuronide, lute-
with the corresponding extract as previously
olin-3-O-b-D-glucuronide and isoorientin
described during 72 h. Controls were test tubes
(Ramesh et al., 1986), have been reported. Also,
containing the cells alone, free of plant extracts.
the triterpenoid friedelin and other steroids, b- The final cell concentrations were determined by
sitosterol and b-sitosterol-b-D-glucoside (Vedan- protein analysis to obtain the effective dose that
tham and Subramanian, 1976; Zeng et al., inhibits 50% growth after the incubation period
1996), have been described, and the chemical (ED50). The values were estimated by means of a
constituents of essential oils obtained from semilog curve derived from extract concentrations
leaves have been analyzed (Pan et al., 1989). (1 mg/ml) plotted against a percentage of viable
In this paper we report several biological ac- cells. Extracts having an ED50 5 20 mg/ml were
tivities (fungicidal, bactericidal, cytotoxic and in- considered active (Villarreal et al., 1992).
3. M.M. Hernandez et al. / Journal of Ethnopharmacology 67 (1999) 37–44
´ 39
2.3. Fungicidal assays inhibition; + + , growth was inhibited above
50%; + , growth was inhibited less than 50%; − ,
Five species of fungi were assayed to determine no inhibition occurred with respect to the control.
the fungicidal activity of the plant extracts: Peni-
cillium sp., Aspergillus fla6us, A. parasiticus, Tri- 2.5. Insecticidal assays
choderma sp. and Fusarium sp. All strains of fungi
were grown in potato dextrose agar (PDA). Plant Insecticidal properties of V. trifolia were tested
extracts dissolved in cyclohexane, acetone, water in the lepidopteran Spodoptera frugiperda (Noctu-
or a mixture of them were added to 5 ml of sterile idae), the most important pest of corn in Mexico,
PDA in a concentration of 500 mg/ml, while con- and also a pest of other crops, such as wheat, soy
trols only contained the solvent where the sample and sorghum, as well as ornamentals (Brown and
was applied. Control and experimental assays Dewhurst, 1975). Larvae of S. frugiperda were
were done by triplicate. Petri dishes were inocu- reared on artificial diet as described (Bell and
lated with 7–10-day-old fungi cultures, and incu- Joachim, 1976). The diet included 0.044% forma-
bated at 28°C during 6 days. Radial mycelial lin, 0.03% acetic acid, and 0.11% choline chloride
growth was monitored every 2 days. Data are (final concentrations) to restrict the growth of
given as percent inhibition as compared to the undesirable microorganisms (Aranda et al., 1996).
controls that reached 100% growth (March et al., Third instar larvae were fed with the plant ex-
1991). tracts contaminating the diet, either mixing the
extracts with the nutrients or spreading a thin
2.4. Bacterial growth inhibition assays layer on top of the congealed diet. Plant extracts
were dissolved in cyclohexane, acetone, water or a
The bacterial growth inhibition assays were per- mixture of them at a concentrations of 10, 100
formed using cultures of Pseudomonas aeruginosa and 1000 mg/ml (mixed with the diet) or 10, 100
(ATCC 9027), Staphylococcus aureus (ATCC and 1000 mg/cm2 (over the surface of the diet),
6538), Shigella sonei (ATCC 11060), Proteus and poured into 24-well polystyrene ELISA
mirabilis, Salmonella typhi (ATCC-CDC-99), as plates. One plate was used per sample dilution
well as the yeast Candida albicans (ATCC 10231). plus one control with the solvents used to dissolve
Bacteria strains were maintained on trypticase soy the extracts. The plates were incubated at 28°C,
agar (TSA) and the yeast on Sabourand’s dex- 60% relative humidity and 16:8 h light:dark pho-
trose agar (SDA). The method was based on toperiod during 7 days. After 7 days, all larvae
conventional disk assays. Plant extracts at 10, 5, were weighed including controls (Kubo, 1991).
2.5, and 1.25 mg/ml were dissolved in 20% DMSO The data were statistically analyzed for a student
in water. However, in order to dissolve the most t-test (Dowdy and Wearden, 1983).
non-polar samples, 20% Tween-20 was added.
The inoculum for each microorganism was pre-
pared from broth culture (108 colony forming 3. Results
units per milliliter, CFU/ml). For testing, 104
CFU were placed by means of a tiny droplet Yields of extracts were as follows (w/w): leaf-
ranging from 5 to 8 mm in diameter, using a hexane, 0.97%; stem-hexane, 0.42%; leaf-DCM,
micropipette calibrated to 2 ml. Controls were 1.95%; stem-DCM, 0.47%; leaf-methanol, 5.61%;
prepared using the same solvents employed to stem-methanol, 2.88%.
dissolve the plant extracts, and as positive control
gentamicin (Pharmacia) was used as reference 3.1. Cytotoxic acti6ity
standard. Each assay was done in duplicate. The
plates were incubated 24 h at 37°C (Villarreal et Table 1 shows the ED50 values for positive
al., 1994). The observed effects were recorded extracts of V. trifolia assayed on four lines of
using pluses as follows: + + +, 100% growth human tumor cells. Hexanic and DCM extracts
4. 40 M.M. Hernandez et al. / Journal of Ethnopharmacology 67 (1999) 37–44
´
Table 1 54% growth inhibition of Fusarium sp. within 4
Cytotoxicity of crude extracts from Vitex trifolia (ED50 mg/ml)
days of the experiment, then, after 6 days, the
Cancer cell linesa Extract inhibition percentage dropped 1.7 times. Growth
inhibition was poor or negligible on the other
Hexane Dichloromethane tested fungi for all extracts (Table 2).
Leaf Stem Leaf Stem
3.3. Bacterial growth inhibition
SQC-1 UISO 15.5 38 2.2 37.1
OVCAR-5 7.6 17.4 2.9 8.5 Table 3 shows the growth inhibition produced
HCT-15 COLAD- 3.6 2.8 B1 1.9
by leaf extracts of V. trifolia toward six species of
CAR
KB 6.0 30.2 1.9 4.1 bacteria (two Gram-positives and four Gram-neg-
atives) and one species of yeast. All extracts com-
a
SQC-1 UISO, cervix carcinoma; OVCAR-5, ovarian can- pletely inhibited the growth of Gram-positive
cer; HCT-15 COLADCAR, colon cancer; KB, nasopharyngeal species (except the methanolic extract assayed at
carcinoma.
the lowest dose). Growth of Gram-negative bacte-
ria were 100% inhibited at 10 mg/ml in all ex-
have shown interesting ED50 values, the DCM tracts, except for S. typhi. No bacterial growth
leaf extract being the most active, with an ED50 was observed when 5 mg/ml of DCM leaf extract
less than 1 mg/ml towards HCT-15 COLADCAR, were assayed, except for S. typhi which was inhib-
which proved to be most sensitive cell line. On the ited over 50% growth. Also, the methanolic leaf
other hand, the SQC-1 UISO cell line was the extract inhibited E. coli and P. mirabilis over 50%
least sensitive. No effects were detected with the growth. At the following dose assayed (2.5 mg/
methanolic extracts of either leaves or stems ml), it was only the DCM leaf extract that par-
(ED50 20 mg/ml). tially inhibited the growth of S. sonei and S.
typhi; the lowest dose of DCM leaf extract that
3.2. Fungicidal acti6ity caused inhibition to C. albicans was 5 mg/ml
(Table 3).
Percentage of growth inhibition of five fungal
species by V. trifolia leaf extracts are shown in 3.4. Insecticidal acti6ity
Table 2. Hexanic leaf extract caused 100% inhibi-
tion of Fusarium sp. within 2 days of growth, later Larvae of S. frugiperda were force-fed from
dropping to 47% at day 4 and to 15% at day 6. contaminated diet with V. trifolia extracts. After 7
On the other hand, the DCM extract exhibited a days, larvae were weighed. A high antifeeding
Table 2
Antifungal activity of crude extracts from Vitex trifolia leaves (percentage of radial mycelial growth inhibition compared to the
control at 2, 4 and 6 days of growth)
Microorganism Percentage inhibition of mycelial growth
Hexane (days) Dichloromethane (days) Methane (days)
2 4 6 2 4 6 2 4 6
Penicillium sp. 22 20 17 27 23 20 0 0 0
Aspegillus fla6us 28 19 15 23 33 29 21 4 4
Aspergillus parasiticus 0 0 0 29 35 23 0 9 4
Tricoderma sp. 20 18 0 21 27 5 0 0 0
Fusarium sp. 100 47 15 54 54 38 31 18 0
6. 42 M.M. Hernandez et al. / Journal of Ethnopharmacology 67 (1999) 37–44
´
triterpenoid friedelin and sitosterol, did not show
cytotoxic values against this same cell line (Zheng,
1994; Wu et al., 1995; Hirobe et al., 1997). None
of the flavonoids that have been already described
in V. trifolia were investigated for their cytotoxic
potential against the cell lines studied in this
investigation, except for KB. There are several
reports which suggest a correlation between the
structures of methoxy flavones as well as 5,7-dihy-
droxy flavones (with substitutions in the B ring),
and with cytotoxic activities (Kupchan et al.,
1971; Woerdenbag et al., 1994). However, other
investigations describe inactive polymethoxy-
Fig. 1. Insecticidal activity of crude dichloromethane (DCM) flavonoids (Kingston et al., 1979), or no relations
extract from Vitex trifolia leaves. Doses were: 1, 0.1, and 0.01 may exist between structures and biological activi-
mg/ml in diet and 1, 0.1 and 0.01 mg/cm2 on diet.Values are
ties (Edwards et al., 1979; Mori et al., 1988).
expressed as mean 9S.D. (n=24). *PB 0.05 and **PB 0.001
significantly different from the control. Moreover, most of these compounds have been
described because of their potent activity against a
activity was recorded in the DCM leaf extract specific cell line, but they have not shown a wider
(Fig. 1). While the larvae had a very low food range of action (Cushman and Nagarathnam,
consumption at the lowest doses assayed, they 1991). It will be desirable to carry out a bioassay
stop feeding at the highest dose employed (1000 guided fractionation of V. trifolia extracts using
mg/cm2), and were dead after a 7-day trial. This the tested cell lines to identify the responsible
behaviour was common when the plant extract compounds for the observed cytotoxic activity,
was layered on the surface of the diet, but when it and to establish whether or not the known
was mixed with the components of the diet, the flavones in this plant are contributing. Two im-
antifeeding effect was recorded only for the portant reasons could support this approach, one
highest dose assayed. is that the most active flavones described for this
plant against KB cells (luteolin and its derivatives)
were isolated from complete crude alcoholic ex-
tracts in which we were unable to detect any
4. Discussion activity, and the other is that important variations
in the flavonoid 6 pattern described for the
Although all prepared extracts from V. trifolia same species have been related to different geo-
showed interesting biological properties, it was graphical distribution of the plants (Nair et al.,
the DCM leaf extract exhibiting the highest cyto- 1975).
toxicity against SQC-1 UISO, OVCAR-5, HCT- It is also noteworthy that the important an-
15 COLADCAR and KB cell lines. These tifeeding activity of the DCM leaves extract
observed actions could probably be attributed to against Spodoptera frugiperda that we observed in
certain secondary compounds in the most apolar this investigation, has been also described with
fraction of the DCM extract that could also be extracts from seeds against larvae of S. litura
present in the hexanic one, in which specific toxic (Hosozawa et al., 1974).
effects against the studied cell lines were also
detected. Some flavonoids already isolated from
this species could have contributed to the general Acknowledgements
toxicity of the plant, i.e. luteolin has shown cyto-
toxicity against KB (ED50 =0.3 1 mg/ml); how- This research was partially supported by Insti-
ever, other flavones, such as artemetin, the tuto Mexicano de Tecnologia del Agua (IMTA)
7. M.M. Hernandez et al. / Journal of Ethnopharmacology 67 (1999) 37–44
´ 43
and Programa de Naciones Unidas pare el De- controlling termite attacks on cassava (Manihot esculenta)
sarrollo (PNUD). We thank Abigail Aguilar with Vitex doniana: a preliminary study (Isoptera). Sociobi-
ology 14 (1), 291 – 297.
(Herbarium of the Centro Medico Nacional,
´ Hebbalkar, D.S., Hebbalkar, G.D., Sharma, R.N., Joshi, V.S.,
IMSS, Mexico City) for authentication of the Bhat, V.S., 1992. Mosquito repellent activity of oils from
plant specimen. We gratefully acknowledge Vitex negundo Linn. leaves. Indian J. Med. Res. 95, 200 –
Daniel Alonso, Socorro Vallejo, Laura Lina and 203.
Victor Navarro for their valuable help in the Hirobe, C., Qiao, Z.S., Takeya, K., Itokawa, H., 1997. Cyto-
biological assays. We also thank the people from toxic flavonoids from Vitex agnus-castus. Phytochemistry
46 (3), 521 – 524.
El Carrizo for their help gathering the plant Hosozawa, S., Kato, N., Munakata, K., Chen, Y.L., 1974.
material. Antifeeding active substances for insects in plants. Agric.
Biol. Chem. 38 (5), 1045 – 1048.
Kingston, D.G., Rao, M.M., Zucker, W.V., 1979. Plant anti-
cancer agents. IX. Constituents of Hyptis tomentosa. J.
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